Calcium gluconate (10 mg Ca++/kg) was administered intravenously over a 2-hour period to 16 adult patients who were evaluated for hypoglycemia. In nine of ten patients with benign or malignant insulinomas (eight proven at operation, and two with positive chemical tests and angiographic localization awaiting operation), significant hypoglycemia and hyperinsulinemia occurred within 60 to 90 minutes after the start of the calcium infusion. Serum proinsulin and C-peptide concentrations increased at the time of the calcium-induced hyperinsulinemia in several patients in whom these parameters were studied. The one individual who did not respond to the calcium infusion was found to have a benign insulinoma. His basal glucose/insulin ratio of 0.64 was the lowest of the insulinoma group and thus his failure to respond to calcium may indicate that his tumor was secreting maximally at the time of the infusion. Following successful removal of the insulinoma, calcium infusion did not result in changes in scrum glucose or insulin concentrations (tested in live patients). In contrast, neither a patient with pathologically documented islet cell hyperplasia, five others with reactive, functional or drug-induced hypoglycemia, nor four healthy volunteers showed any changes in circulating glucose or insulin levels while receiving calcium intravenously. Calcium infusion is a safe, rapid and effective provocative test for the diagnosis of insulin-secreting, islet cell tumors of the pancreas.